Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of Distal Arthrogryposis

Abstract

Mechanotransduction, the pathway by which mechanical forces are translated to biological signals, plays important but poorly characterized roles in physiology. PIEZOs are recently identified, widely expressed, mechanically activated ion channels that are hypothesized to play a role in mechanotransduction in mammals. Here, we describe two distinct PIEZO2 mutations in patients with a subtype of Distal Arthrogryposis Type 5 characterized by generalized autosomal dominant contractures with limited eye movements, restrictive lung disease, and variable absence of cruciate knee ligaments. Electrophysiological studies reveal that the two PIEZO2 mutations affect biophysical properties related to channel inactivation: both E2727del and I802F mutations cause the PIEZO2-dependent, mechanically activated currents to recover faster from inactivation, while E2727del also causes a slowing of inactivation. Both types of changes in kinetics result in increased channel activity in response to a given mechanical stimulus, suggesting that Distal Arthrogryposis Type 5 can be caused by gain-of-function mutations in PIEZO2. We further show that overexpression of mutated PIEZO2 cDNAs does not cause constitutive activity or toxicity to cells, indicating that the observed phenotype is likely due to a mechanotransduction defect. Our studies identify a type of channelopathy and link the dysfunction of mechanically activated ion channels to developmental malformations and joint contractures.

Fig. 1.

Dysmorphic features of DA5 individuals. (A) Infant photographs of individual 2 showing deep-set small eyes with mild ptosis, restricted shoulder abduction, and flexion contractures in the knees. Deep skin dimples can be seen on the shoulder (red circle) and sternum. (B) The DA of individual 2, 3, and 1 (from top to bottom). Note flexion contractures of the interphalangeal joints, increasing in severity from thumb to little finger. (C) Retinal photograph of individual 3 shows pigmented macular striae. This was not found in individual 1. (D) Flow volume loop of individual 3 showing markedly reduced total lung capacity (∼1 L) and restricted flow dynamics during both inspiration and expiration. (E) Hydrophobicity plot of human PIEZO2 showing Kyte–Doolittle hydrophobicity analysis (19-residue window) done with ProtScale program (Expasy). Triangles indicate the position of I802F and E2727del mutations.

Fig. 2.

E2727del PIEZO2 channels display slower inactivation kinetics compared with wild type. (A) Representative traces of MA inward currents at –80 mV in cells transfected with the indicated constructs and subjected to a series of mechanical steps in 1 µm increments. (B) Average maximal current amplitude of MA inward currents at –80 mV. The number of cells tested is shown above bars. (C) Averaged current-voltage relationships of MA currents in cells transfected with hPiezo2 (n = 7 cells), I802F (n = 8 cells), or E2727del (n = 8 cells). The insets show representative MA currents evoked at holding potentials ranging from –80 to +80 mV; inset scale bars are 1 nA and 100 ms. (D) Representative traces of MA currents elicited at –80 and +80 mV holding potentials. Traces were normalized to the peak current, and blue, red, and green dashed lines represent fits of inactivation with a monoexponential equation of human PIEZO2, I802F, and E2727del currents, respectively. (E) Time-constant of inactivation (tau) of PIEZO2 (black dots, n = 7 cells), I802F (red dots, n = 8 cells), and E2727del (green dots, n = 8 cells) currents at different holding potentials. Dots and bars represent mean ± SEM. ns, not statistically different; **P < 0.01; ***P < 0.001; Mann–Whitney test.

Fig. 3.

E2727del and I802F PIEZO2 channels recover faster from inactivation compared with wild type. (A) Typical recording traces of PIEZO2, I802F, and E2727del MA currents recorded at –80 mV. The protocol depicted on the top consists of two consecutive mechanical stimulation steps separated by increasing delay and aims at testing recovery from inactivation. The traces for each construct represent superimposition of multiple test-control stimulation pairings at different delta-t intervals. The test current traces are each normalized to the peak of control currents. Peak of test currents are fitted with a monoexponential equation. (B) Average of test peak current/control peak current for PIEZO2 (black, n = 8 cells), I802F (red, n = 7 cells), and E2727del (green, n = 7 cells) are fitted with monoexponential equation. (C) Average of time-constant (tau) of recovery from inactivation determined for each individual cell exemplified in A. The number of cells tested is shown above bars. Dots and bars represent mean ± SEM. **P < 0.01; ***P < 0.001; Mann–Whitney test.